Porin activity and sequence analysis of a 31-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp1).

We have recently reported the isolation and purification of the Treponema pallidum outer membrane and the identification of its rare protein constituents, including a 31-kDa protein markedly enriched in the outer membrane preparation (D.R. Blanco, K. Reimann, J. Skare, C.I. Champion, D. Foley, M. M. Exner, R. E. W. Hancock, J. N. Miller, and M. A. Lovett, J. Bacteriol. 176:6088-6099, 1994). In this study, we report the cloning, sequencing, and expression of the structural gene which encodes the 31-kDa outer membrane protein, designated Tromp1. The deduced amino acid sequence from the tromp1 gene sequence encodes a 318-amino-acid polypeptide with a putative 40-amino-acid signal peptide. Processing of Tromp1 results in a mature protein with a predicted molecular mass of 30,415 Da and a calculated pI of 6.6. Secondary-structure predictions identified repeated stretches of amphipathic beta-sheets typical of outer membrane protein membrane-spanning sequences. A topological model of Tromp1 containing 14 transmembrane segments is proposed. Specific antiserum against a recombinant Tromp1 fusion protein was generated and was used to identify native Tromp1 in cellular fractionation. Upon Triton X-114 extraction and phase separation of T. pallidum, the 31-kDa Tromp1 protein was detected in the detergent-phase fraction but not in the protoplasmic cylinder or aqueousphase fractions, consistent with a hydrophobic outer membrane protein. Anti-Tromp1 antiserum was also used to identify native Tromp1 purified from whole T. pallidum by Triton X-100 solubilization followed by nondenaturing isoelectric focusing. Reconstitution of purified Tromp1 into planar lipid bilayers showed porin activity based on the measured single channel conductanes of 0.15 and 0.7 nS in 1 M KCl. These findings demonstrate that Tromp1 is a transmembrane outer membrane porin protein of T. pallidum.     

Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2).

In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type I signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pI of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum.     

Renaturation of recombinant Treponema pallidum rare outer membrane protein 1 into a trimeric, hydrophobic, and porin-active conformation

We have previously observed that while native Treponema pallidum rare outer membrane protein 1 (Tromp1) is hydrophobic and has porin activity, recombinant forms of Tromp1 do not possess these properties. In this study we show that these properties are determined by conformation and can be replicated by proper renaturation of recombinant Tromp1. Native Tromp1, but not the 47-kDa lipoprotein, extracted from whole organisms by using Triton X-114, was found to lose hydrophobicity after treatment in 8 M urea, indicating that Tromp1's hydrophobicity is conformation dependent. Native Tromp1 was purified from 0.1% Triton X-100 extracts of whole organisms by fast-performance liquid chromatography (FPLC) and shown to have porin activity in planar lipid bilayers. Cross-linking studies of purified native Tromp1 with an 11 A cross-linking agent showed oligomeric forms consistent with dimers and trimers. For renaturation studies of recombinant Tromp1 (rTromp1), a 31,109-Da signal-less construct was expressed in Escherichia coli and purified by FPLC. FPLC-purified rTromp1 was denatured in 8 M urea and then renatured in the presence of 0.5% Zwittergent 3,14 during dialysis to remove the urea. Renatured rTromp1 was passed through a Sephacryl S-300 gel exclusion column previously calibrated with known molecular weight standards. While all nonrenatured rTromp1 eluted from the column at approximately the position of the carbonic anhydrase protein standard (29 kDa), all renatured rTromp1 eluted at the position of the phosphorylase b protein standard (97 kDa), suggesting a trimeric conformation. Trimerization was confirmed by using an 11 A cross-linking agent which showed both dimers and trimers similar to that of native Tromp1. Triton X-114 phase separations showed that all of renatured rTromp1, but none of nonrenatured rTromp1, phase separated exclusively into the hydrophobic detergent phase, similar to native Tromp1. Circular dichroism of nonrenatured and renatured rTromp1 showed a marked loss in alpha-helical secondary structure of renatured rTromp1 compared to the nonrenatured form. Finally, renatured rTromp1, but not the nonrenatured form, showed porin activity in planar liquid bilayers. These results demonstrate that proper folding of rTromp1 results in a trimeric, hydrophobic, and porin-active conformation similar to that of the native protein.     

Isolation of the outer membranes from Treponema pallidum and Treponema vincentii.

The outer membranes from Treponema pallidum subsp. pallidum and Treponema vincentii were isolated by a novel method. Purified outer membranes from T. pallidum and T. vincentii following sucrose gradient centrifugation banded at 7 and 31% (wt/wt) sucrose, respectively. Freeze fracture electron microscopy of purified membrane vesicles from T. pallidum and T. vincentii revealed an extremely low density of protein particles; the particle density of T. pallidum was approximately six times less than that of T. vincentii. The great majority of T. vincentii lipopolysaccharide was found in the outer membrane preparation. The T. vincentii outer membrane also contained proteins of 55 and 65 kDa. 125I-penicillin V labeling demonstrated that t. pallidum penicillin-binding proteins were found exclusively with the protoplasmic cylinders and were not detectable with purified outer membrane material, indicating the absence of inner membrane contamination. Isolated T. pallidum outer membrane was devoid of the 19-kDa 4D protein and the normally abundant 47-kDa lipoprotein known to be associated with the cytoplasmic membrane; only trace amounts of the periplasmic endoflagella were detected. Proteins associated with the T. pallidum outer membrane were identified by one- and two-dimensional electrophoretic analysis using gold staining and immunoblotting. Small amounts of strongly antigenic 17- and 45-kDa proteins were detected and shown to correspond to previously identified lipoproteins which are found principally with the cytoplasmic membrane. Less antigenic proteins of 65, 31 (acidic pI), 31 (basic pI), and 28 kDa were identified. Compared with whole-organism preparations, the 65- and the more basic 31-kDa proteins were found to be highly enriched in the outer membrane preparation, indicating that they may represent the T. pallidum rare outer membrane proteins. Reconstitution of solubilized T. pallidum outer membrane into lipid bilayer membranes revealed porin activity with two estimated channel diameters of 0.35 and 0.68 nm based on the measured single-channel conductances in 1 M KCl of 0.40 and 0.76 nS, respectively.     

Stable expression of meningococcal class 1 protein in an antigenically reactive form in outer membranes of Escherichia coli

The entire gene encoding the class 1 outer membrane protein of Neisseria meningitidis is located on a 2.2kb fragment, obtained on digestion of chromosomal DNA with Xbal. This Xbal fragment from strain MC50 (subtype P1-16), which had previously been cloned in bacteriophage M13, has been transferred to the plasmid vector pMTL20. The resulting plasmid (pPORA100) was propagated in Escherichia coli (JM109) and cell lysates were subjected to SDS-PAGE. Western blotting with anti-class 1 protein antibodies revealed constitutive expression of a protein of 41 kD, corresponding to the class 1 protein of the parent meningococcal strain, which was absent in the E. coli control. Fractionation of E. coli cells carrying the recombinant plasmid revealed that the protein was exclusively located in the outer membrane, and N-terminal amino acid analysis of the expressed protein revealed that normal processing of the signal peptide had occurred. Immuno-gold electron microscopy showed that the protective epitope recognized by a P1-16 subtype-specific monoclonal antibody was exposed in an antigenically reactive form on the surface of E. coli cells carrying plasmid pPORA100. In contrast, expression in E. coli of a second plasmid (pPORA104) lacking the coding sequence for the first 15 amino acids of the signal peptide resulted in accumulation of recombinant class 1 protein only in the cytoplasm of the cells. Thus the presence of the meningococcal signal sequence ensures expression of this meningococcal porin protein in an antigenically native conformation in outer membranes of E. coli, while its absence results in expression of a soluble protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Demonstration by Mass Spectrometry that Purified Native Treponema pallidum Rare Outer Membrane Protein 1 (Tromp1) Has a Cleaved Signal Peptide

Purified native Tromp1 was subjected to mass spectrometric analysis in order to determine conclusively whether this protein possesses a cleaved or uncleaved signal peptide. The molecular masses of Tromp1, three Treponema pallidum lipoproteins, and a bovine serum albumin (BSA) control were determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The molecular masses of all of the T. pallidum lipoproteins and BSA were within 0.7% of their respective calculated masses. The molecular mass of Tromp1 was 31,510 Da, which is consistent with a signal-less form of Tromp1, given a calculated mass of unprocessed Tromp1 of 33, 571 Da, a difference of 2,061 Da (a 6.5% difference). Purified native Tromp1 was also subjected to MALDI-TOF analysis in comparison to recombinant Tromp1 following cyanogen bromide cleavage, which further confirmed the identity of Tromp1 and showed that native Tromp1 was not degraded at the carboxy terminus. These studies confirm that Tromp1 is processed and does not contain an uncleaved signal peptide as previously reported.     

Isolation of an ompC-like outer membrane protein gene from Salmonella typhi

We have isolated the structural gene for an outer membrane protein of Salmonella typhi, from a genomic library constructed in bacteriophage lambda 1059, using the Escherichia coli ompC gene as a heterologous probe. E. coli ompC codes for an outer membrane pore protein (porin) that is induced preferentially at high osmolarity and high temperature. The S. typhi ompC-like gene was subcloned in pBR322 and introduced into E. coli HB101 and into P678-54, a minicell-producing strain. In both strains it expressed a 38.5-kDa protein, which was incorporated into the outer membrane envelope and comigrated with an S. typhi outer membrane protein which was expressed both at low and high osmolarity in vivo.     

Outer membrane porin proteins F, P, and D1 of Pseudomonas aeruginosa and PhoE of Escherichia coli: chemical cross-linking to reveal native oligomers.

Native oligomers of three Pseudomonas aeruginosa outer membrane porin proteins and one Escherichia coli porin were demonstrated by using a chemical cross-linking technique. P. aeruginosa protein F, the major constitutive outer membrane porin, was cross-linked to dimers in outer membrane and whole-cell cross-linking experiments. Purified preparations of P. aeruginosa proteins F, D1 (glucose induced), and P (phosphate starvation induced) and E. coli protein PhoE (Ic) were also cross-linked to reveal dimers and trimers upon two-dimensional sodium dodecyl sulfate-polyacrylamide electrophoretic analysis. Cross-linking of protein F was abolished by pretreatment of the protein with sodium dodecyl sulfate, indicating that the cross-linked products were due to native associations in the outer membrane.     

Verification of the protein in the outer membrane of Bdellovibrio bacteriovorus as the OmpF protein of its Escherichia coli prey.

Two research groups showed that several Bdellovibrio strains incorporated into their outer membranes intact OmpF porin proteins derived from their Escherichia coli prey. These results could not be reproduced by another group using Bdellovibrio bacteriovorus 109J. They showed that a major protein appearing in the Bdellovibrio Triton X-100-insoluble outer membrane was coded for by the bdellovibrios. We reconciled these results by examining the strain used by this group and by reviving a freeze-dried culture of strain 109J which had been stored for almost 9 years. B. bacteriovorus 109J failed to acquire substantial amounts of the OmpF protein from E. coli ML35, and a protein coded for by the bdellovibrios was expressed in its place. However, B. bacteriovorus 109J incorporated the OmpF protein from rough K-12 strains of E. coli, and the revived 9-year-old culture of B. bacteriovorus 109J incorporated more of the OmpF protein from the smooth E. coli ML35 than did its contemporary counterpart. The protein isolated from the outer membrane of the bdellovibrios was identified as the OmpF protein of E. coli by its protease peptide profile on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western blot analysis. This confirmed that bdellovibrios relocalize outer membrane proteins from their prey, but relocalization may be an unstable trait which can be influenced by the prey.     

Molecular and functional characterisation of the Serratia marcescens outer membrane protein Omp1

Serratia marcescens outer membrane contains three different general diffusion porins: Omp1, Omp2 and Omp3. Omp1 was cloned and sequenced and it shows a great homology to the family of outer membrane porins that comprises the general porins of enteric bacteria. The gene for Omp1 was transferred into an expression plasmid and was expressed in Escherichia coli UH302 (E. coli UH302 pOM100), a porin deficient strain. Its expression confers a higher susceptibility towards different antibiotics to this strain. Omp1 was purified to homogeneity from outer membrane of E. coli UH302 pOM100. Reconstitution of the purified protein into black lipid bilayers demonstrated that it is a channel-forming component with a single-channel conductance of approximately 2 nS in 1 M KCl similar to that of other porins from enteric bacteria. Omp1 is slightly cation-selective. Its homology to already crystallised members of the family of enteric porins whose three-dimensional-structures are known and allowed the design of a topology model for Omp1. The charge distribution within a porin monomer is similar as in other general diffusion pores. The positively charged amino acids localised at the beta-strands opposite the external loop L3, which restrict the pore diameter in the porin monomer.     

Isolation and characterization of a conserved porin protein from Helicobacter pylori.

Helicobacter pylori is a causative agent of gastritis in humans and is correlated with gastric ulcer formation. Infections with this bacterium have proven difficult to treat with antimicrobial agents. To better understand how this bacterium transports compounds such as antimicrobial agents across its outer membrane, identification of porin proteins is important. We have recently identified a family of H. pylori porins (HopA to HopD) (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567-1572, 1995). Here, we report on an unrelated porin species (HopE) from this bacterium. This protein had a apparent molecular mass of 31 kDa and was seen to form 50- and 90-kDa aggregates that were designated putative dimeric and trimeric forms, respectively. The protein was purified to homogeneity and, with a model planar lipid membrane system, was shown to act as a nonselective pore with a single channel conductance in 1.0 M KCl of 1.5 nS, similarly to other bacterial nonspecific porins. An internal peptide sequence of HopE shared homology with the P2 porin of Haemophilus influenzae. HopE was also shown to be antigenic in vivo as assessed by sera taken from H. pylori-infected individuals and was immunologically conserved with both patient sera and specific monoclonal antibodies. From these data, it appears that HopE is a major nonselective porin of H. pylori. The implications of these findings are discussed.     

Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp.

Pathogenic Leptospira spp. are spirochetes that have a low transmembrane outer membrane protein content relative to that of enteric gram-negative bacteria. In a previous study we identified a 31-kDa surface protein that was present in strains of Leptospira alstoni in amounts which correlated with the outer membrane particle density observed by freeze fracture electron microscopy (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). The N-terminal amino acid sequence was used to design a pair of oligonucleotides which were utilized to screen a lambda ZAP II library containing EcoRI fragments of L. alstoni DNA. A 2.5-kb DNA fragment which contained the entire structural ompL1 gene was identified. The structural gene deduced from the sequence of this DNA fragment would encode a 320-amino-acid polypeptide with a 24-amino-acid leader peptide and a leader peptidase I cleavage site. Processing of OmpL1 results in a mature protein with a predicted molecular mass of 31,113 Da. Secondary-structure prediction identified repeated stretches of amphipathic beta-sheets typical of outer membrane protein membrane-spanning sequences. A topological model of OmpL1 containing 10 transmembrane segments is suggested. A recombinant OmpL1 fusion protein was expressed in Escherichia coli in order to immunize rabbits with the purified protein. Upon Triton X-114 extraction of L. alstoni and phase separation, anti-OmpL1 antiserum recognized a single band on immunoblots of the hydrophobic detergent fraction which was not present in the hydrophilic aqueous fraction. Immunoelectron microscopy with anti-OmpL1 antiserum demonstrates binding to the surface of intact L. alstoni. DNA hybridization studies indicate that the ompL1 gene is present in a single copy in all pathogenic Leptospira species that have been tested and is absent in nonpathogenic Leptospira species. OmpL1 may be the first spirochetal transmembrane outer membrane protein for which the structural gene has been cloned and sequenced.     

Identification of a novel membrane-associated gene product that suppresses toxicity of a TrfA peptide from plasmid RK2 and its relationship to the DnaA host initiation protein

The toxicity of a peptide derived from the amino-terminal portion of 33-kDa TrfA, one of the initiation proteins encoded by the broad-host-range plasmid RK2, was suppressed by a host protein related to DnaA, the initiation protein of Escherichia coli. The newly identified 28.4-kDa protein, termed a DnaA paralog (Dp) because it is similar to a region of DnaA but likely has a different function in initiation of plasmid RK2 replication, interacts physically with the 33-kDa TrfA initiation protein, including the initiation-active monomeric form. The Dp has a cellular distribution similar to that of the 33-kDa TrfA initiation protein, being found primarily in the inner membrane fraction, with lesser amounts detected in the outer membrane fraction and almost none in the soluble fraction of E. coli. Maintenance and inner membrane-associated replication of plasmid RK2 were enhanced in a Dp knockout strain and inhibited in strains containing extra copies of the Dp gene or in membrane extracts to which a tagged form of Dp was added. Recently, the Dp was independently shown to help prevent overinitiation in E. coli and was termed Hda (S. Kato and T. Katayama, EMBO J. 20:4253-4262, 2001).     

Ion selectivity reversal and induction of voltage-gating by site-directed mutations in the Paracoccus denitrificans porin

The porin from Paracoccus denitrificans, a slightly anion specific outer membrane pore protein, was expressed in Escherichia coli, isolated from inclusion bodies, and refolded in the presence of urea and detergents. The purified recombinant protein was reconstituted into black lipid bilayer membranes and showed no difference in its functional properties in comparison to the native porin isolated from P.denitrificans membranes. To investigate the molecular basis of its ion selectivity and voltage-gating, a series of site-directed mutants was constructed, comprising acidic residues located on the third extracellular loop (L3), which forms the constriction zone of the channel, and basic residues along the opposing barrel wall. Measurements using zero-current membrane potentials indicated that the selectivity changed drastically from a slight anion to a distinct cation selectivity with the exchange of residues R29 and R31 by glutamate, whereas replacements on the L3 loop went largely unaffected. However, when assaying the voltage-dependent closure of channels, only mutations located on the L3 loop showed an effect, in contrast to the voltage-independent recombinant and native Paracoccus porin.     

Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli.

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.     

Pore formation and mitogenicity in blood cells by the class 2 protein of Neisseria meningitidis.

The class 2 outer membrane protein (MIEP) of Neisseria meningitidis has recently been shown to be mitogenic for lymphocytes (Liu, M.A., Friedman, A., Tai, J., Martinez, D., Deck, R. R., Hawe, L. A., Shieh, J. T.-C., Jenkins, T. D., Donnelly, J. J., and Oliff, A. I. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4633-4637. In this study, a possible connection between MIEP's mitogenicity and its possible action as a porin was investigated. MIEP, purified from the bacterial outer membrane protein complex under denaturing conditions, caused a modest but specific release of ions (86Rb) from both erythrocytes and lymphocytes, ultimately resulting in cell lysis. The dose-response of MIEP on erythrocyte lysis was qualitatively similar to a known porin (protein I from Neisseria gonorrhoeae) but was much less efficient. Induction or preservation of native structure in MIEP increased pore formation, resulting in levels comparable to that of the protein I porin. These observations suggest that native MIEP, free of the other outer membrane proteins of Neisseria meningitidis, can efficiently form pores in cells, but that denatured MIEP is variably and marginally effective. However, pore formation by MIEP was not related to its mitogenicity in lymphocytes, based on: (i) native MIEP was not mitogenic; (ii) denatured MIEP was highly mitogenic; and (iii) denatured MIEP was mitogenic at concentrations below the threshold level for pore formation. Therefore, mitogenicity is dependent upon MIEP being in a denatured, monomeric state and is masked by native conformation.     

Expression of the Chlamydia trachomatis major outer membrane protein-encoding gene in Escherichia coli: role of the 3' end in mRNA stability

The major outer membrane protein (MOMP)-encoding gene (omp1) of Chlamydia trachomatis has been cloned into Escherichia coli and partially sequenced. This recombinant gene expresses a full-length 40-kDa product, which is recognized by a monoclonal antibody directed against the species-specific epitope of MOMP. The recombinant omp1 is expressed in either insertion orientation, indicating that it utilizes its own promoter system. The endogenous omp1 promoter possesses a relatively low activity despite the high level of MOMP expression. Deletion of a 520-bp fragment at the 3' end encoding 39 amino acids (aa) at the C terminus and the remainder of the noncoding region leads to a significant decrease in mRNA stability and loss of protein synthesis. When the MOMP-encoding plasmid was introduced into E. coli minicells, it expressed 40- and 43-kDa proteins; however, inhibition of post-translational processing by ethanol revealed only a 43-kDa protein. These data indicate that the unprocessed omp1 gene product contains a 22-aa leader sequence which is cleaved during translocation to the outer membrane, to yield a processed 40-kDa protein. The recombinant MOMP was localized to the outer membrane E. coli fraction, comparable to the location of the native C. trachomatis protein.     

Sequence analysis and characterization of a 40-kilodalton Borrelia hermsii glycerophosphodiester phosphodiesterase homolog.

We report the purification, molecular cloning, and characterization of a 40-kDa glycerophosphodiester phosphodiesterase homolog from Borrelia hermsii. The 40-kDa protein was solubilized from whole organisms with 0.1% Triton X-100, phase partitioned into the Triton X-114 detergent phase, and purified by fast-performance liquid chromatography (FPLC). The gene encoding the 40-kDa protein was cloned from a B. hermsii chromosomal DNA lambda EXlox expression library and identified by using affinity antibodies generated against the purified native protein. The deduced amino acid sequence included a 20-amino-acid signal peptide encoding a putative leader peptidase II cleavage site, indicating that the 40-kDa protein was a lipoprotein. Based on significant homology (31 to 52% identity) of the 40-kDa protein to glycerophosphodiester phosphodiesterases of Escherichia coli (GlpQ), Bacillus subtilis (GlpQ), and Haemophilus influenzae (Hpd; protein D), we have designated this B. hermsii 40-kDa lipoprotein a glycerophosphodiester phosphodiesterase (Gpd) homolog, the first B. hermsii lipoprotein to have a putative functional assignment. A nonlipidated form of the Gpd homolog was overproduced as a fusion protein in E. coli BL21(DE3)(pLysE) and was used to immunize rabbits to generate specific antiserum. Immunoblot analysis with anti-Gpd serum recognized recombinant H. influenzae protein D, and conversely, antiserum to H. influenzae protein D recognized recombinant B. hermsii Gpd (rGpd), indicating antigenic conservation between these proteins. Antiserum to rGpd also identified native Gpd as a constituent of purified outer membrane vesicles prepared from B. hermsii. Screening of other pathogenic spirochetes with anti-rGpd serum revealed the presence of antigenically related proteins in Borrelia burgdorferi, Treponema pallidum, and Leptospira kirschneri. Further sequence analysis both upstream and downstream of the Gpd homolog showed additional homologs of glycerol metabolism, including a glycerol-3-phosphate transporter (GlpT), a glycerol-3-phosphate dehydrogenase (GlpD), and a thioredoxin reductase (TrxB).     

Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the Asd+ vector pYA292, and the construct was introduced into the avirulent delta cya delta crp delta asd S. typhimurium chi 3987 for oral immunization of birds. The gene encoding the 21-kDa protein was expressed equivalently in B. avium 197, delta asd E. coli chi 6097, and S. typhimurium chi 3987 and was localized primarily in the cytoplasmic membrane and outer membrane. In preliminary studies on oral inoculation of turkey poults with S. typhimurium chi 3987 expressing the gene encoding the B. avium 21-kDa protein, it was determined that a single dose of the recombinant Salmonella vaccine failed to elicit serum antibodies against the 21-kDa protein and challenge with wild-type B. avium 197 resulted in colonization of the trachea and thymus with B. avium 197.     

Raising antibodies against OprD, an outer membrane protein of Pseudomonas aeruginosa using translational fusions to MalE.

OprD is an outer membrane porin of Pseudomonas aeruginosa that mediates uptake of basic amino acids, peptides as well as carbapenem antibiotics. Polyclonal antibodies were raised against the OprD porin by creating protein fusions between the Escherichia coli maltose binding protein and four OprD fragments. These were expressed in E. coli and shown to be exported to the periplasm. The fusion proteins were purified by amylose affinity chromatography and used to immunize rabbits intramuscularly. We established that MalE fusions to OprD fragments retain maltose and amylose binding activities in vivo and in vitro, confirming proper folding of the MalE domain of hybrid proteins. Furthermore, we demonstrate that this strategy can be used to obtain specific antibodies against bacterial outer membrane proteins (OMPs).